SignificanceDivision-of-focal-plane Stokes polarimetry is emerging as a powerful tool for the microstructural characterization of soft tissues. How individual extracellular matrix (ECM) properties influence polarimetric signals in reflectance or transmission modes of quantitative polarized light imaging (QPLI) is not well understood.AimWe aimed to investigate how ECM properties affect outcomes obtained from division-of-focal-plane polarimetric imaging in reflectance or transmission modes.ApproachTunable collagen gel phantoms were used to modulate ECM properties of anisotropy, collagen density, crosslinking, and absorber density; the effects of degree of linear polarization (DoLP) and angle of polarization (AoP) on polarimetry outcomes were assessed. A model biological tissue (i.e., bovine tendon) was similarly imaged and evaluated using both reflectance and transmission modes.ResultsReflectance QPLI resulted in decreased DoLP compared with transmission mode. A 90 deg shift in AoP was observed between modes but yielded similar spatial patterns. Collagen density had the largest effect on outcomes besides anisotropy in both imaging modes.ConclusionsBoth imaging modes were sufficiently sensitive to detect structural anisotropy differences in gels of varying fiber alignment. Conclusions drawn from phantom experiments should carry over when interpreting data from more complex tissues and can help provide context for interpretation of other Stokes polarimetry data.
Polarimetry has long been used to investigate fiber anisotropy of biological soft tissues, either independently or together with other imaging methods, as it leverages the natural birefringence of collagen. Despite widespread usage, previous studies of soft tissues contain ambiguous interpretations of data gathered from polarized light-based techniques. To date, there has not yet been a systematic assessment of how individual extracellular matrix (ECM) properties influence the polarization of light, which limits the ability to correctly interpret data from these techniques in some applications. To probe the effect of various ECM properties on polarized light, we used a tunable hydrogel system to vary the collagen density, crosslinking density, and absorber concentration. Samples were imaged using quantitative polarized light imaging (QPLI), which uses circularly polarized incident light and a division of focal plane polarimeter. QPLI was performed in both reflectance and transmission modes. The average degree of linear polarization (AVG DoLP; i.e., strength of alignment) and standard deviation of the angle of polarization (STD AoP; i.e., uniformity of alignment) were calculated for each hydrogel. Increasing collagen density resulted in the most pronounced changes, where AVG DoLP and STD AoP increased for reflectance and transmission mode, likely due to the increased concentration of birefringent material. Crosslinking only caused a modest increase on AVG DoLP in transmission mode but decrease in STD AoP in reflectance mode, likely due to the small length scale of the crosslink relative to the fibers. Alteration of transmissivity resulted in changes mainly in reflectance mode, where multiple scattering was more pronounced. Results will help improve data interpretation and experimental control when using polarized light to image biological soft tissues.
Biomechanical function of musculoskeletal soft tissues is dictated by their hierarchically organized collagen extracellular matrices. Our lab has used quantitative polarized light imaging (QPLI) to evaluate dynamic collagen fiber alignment in soft tissues. However, thinning of tissue for light transmission precludes the use of QPLI in more physiologically relevant scenarios. Reflectance mode QPLI could allow for in situ analysis of collagen microstructure. In this study, signal obtained via reflectance and transmission mode QPLI of engineered soft tissue analogs with prescribed collagen fiber alignment were compared in order to understand differences between modes and to provide context for further orthopedic applications.
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